Genetic identification and purification of the respiratory NADH dehydrogenase of Escherichia coli.
Escherichia coli membrane particles were solubilized with potassium cholate. An NADH:ubiquinone oxidoreductase was resolved by hydroxylapatite chromatography of the solubilized material. This enzyme has been identified as the respiratory NADH dehydrogenase since it is absent in chromatograms of solubilized material from an ndh mutant strain. Such mutants lack membrane-bound NADH oxidase activity and have previously been shown to have an inactive NADH dehydrogenase complex [Young, I. G., & Wallace, B. J. (1976) Biochim. Biophys. Acta 449, 376-385]. The respiratory NADH dehydrogenase was amplified 50- to 100-fold in vivo by using multicopy plasmid vectors carrying the ndh gene and then purified to homogeneity on hydroxylapatite. Hydroxylapatite chromatography of cholate-solubilized material from genetically amplified strains purified the enzyme approximately 800- to 100-fold relatively to the activity in wild-type membranes. By use of a large-scale purification procedure, 50-100 mg of protein with a specific activity of 500-600 mumol of reduced nicotinamide adenine dinucleotide oxidized min-1 mg-1 at pH 7.5, 30 degrees C, was obtained. Sodium dodecyl sulfate gel electrophoresis of the purified enzyme showed that the enzyme consists of a single polypeptide with an apparent Mr of 45 000.
D002462:Cell Membrane / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology*; D005796:Genes; D005816:Genetic Complementation Test; D008970:Molecular Weight; D009154:Mutation; D016660:NAD(P)H Dehydrogenase (Quinone); D009247:NADH, NADPH Oxidoreductases / Q000235:genetics*; D010957:Plasmids; D011808:Quinone Reductases / Q000235:genetics* / Q000302:isolation & purification
D009247:NADH, NADPH Oxidoreductases; D016660:NAD(P)H Dehydrogenase (Quinone); D011808:Quinone Reductases